Construction Of TbpB-defective Mutants Of Haemophilus Parasuis And The Typing By PCR-RFLP Analysis Of The TbpB Gene

Haemophilus parasuis is a non-motile, nicotinamide adenine dinucletide(NAD)-dependent, Gram-negative rod of the family Pasteurellaceae (belonging to theγ subclass of the class Proteobacteria). This species is the causative agent responsiblefor Gl sser’s disease. It is an emerging challenge in the pig-rearing industryworldwide. Although significant studies on HPS have been reported as well as severalvirulence factors have been screened, the pathogenesis is yet to be understood.Amongout membrance protein （Omps）, transferrin-binding proteins (tbps) in othergram-negative organisms have been considered as important targets for thedevelopment of attenuated live vaccines because an impairment of iron uptakemechanisms is likely to reduse virulence.Although PCR-RFLP of tbpA have already been characterized in H.parasuis,information regarding tbpB is scarce in this species. In the current study, tbpB gene ofthe15H.parasuis reference strains was used for genetyping by PCR-RFLP. Primersbased on the nucleotide sequence from H.parasuis SH0165strains (GenBank:CP001321.1), and DNA sequencing of the15H.parasuis tbpB genes were carried out.Using molecular biology software-Vector NTI, the transferring-binding proteins-tbpBwas analyzed with AluⅠ，AvaⅡ and RsaⅠ endonucleases. Reference serovarsproduced11different genotypes. GenotypⅠe(AAA) included serovar1,3,15andgenotypeⅡ(BBB) was represented by serovar2,9,11. Isolates had10genotypes,and6of those was new. The fact suggests heterogeneity of H.parasuis isolates and thatPCR-RFLP is an useful method for studing the epidemiology of outbreaks ofGlasser’s disease, therefore, further validation with more field strains is needed.In the current study, H.parasuis tbpB gene’s exogenous deletion fragment wasconstructed through inverse PCR technique. Then, the deletion fragment was insertedinto suicide plasmid pEMOC2to get pEM-△tbpB, which can stablely duplicated inE.coliβ2155. E.coliβ2155was used as donor cell and H.parasuis serotype V (Nagasaki strain) was used as recipient top perform the transconjugation. Afterprimary screening on the plate, the Chloramphenicol-resistant (CmR) and sucrosesensitivity (SucS) transconjugants were then identified by PCR to make sure whetherthe suicide plamide was successfully inserted. Then, copy the interestedtranscongugats to TSA plates containing10%(w/v) sucrose to be incubated for24h toproceed the Chloramphenicol-resistant (CmR) and sucrose sensitivity (SucS) screening,prior to use PCR technique to make sure the Chloramphenicol-sensitivy (CmS) andsucrose-resistant (SucS) transconjugats are acquired, which contains the deletionfragment, but without the suicide plasmid. The results will be useful for identifyinggene function and virelence-associated genes in bacterial pathogens, and developing alive attenuated genetic engineering vaccine of H.parasuis.